The objective of the proposed research is to identify which of several different mechanisms of calcium release from the sarcoplasmic reticulum (SR) contribute to excitation-contraction coupling in vertebrate skeletal muscle. A mechanistic approach will be utilized involving the use of pharmacologic agents as tools. Identified pharmacologic agents will be introduced into muscle fibers in order to test which mechanisms for Ca2+ release are physiologically important. These determinations will utilize skeletal muscle fibers voltage clamped in a triple Vaseline gap chamber. The experiments will involve optical determinations of contractile thresholds and myoplasmic Ca2+ transients measured using metallochromic indicator dyes. The choice of agents used for the muscle fiber studies will be made on the basis of their effects on different mechanisms of calcium release from isolated SR. Specific inhibitors will be identified for SR calcium release induced by Ca2+, alkalinization, SR depolarization, inositol 1,4,5 triphosphate, quinine and other agents. These experiments will involve measurements of Ca 2+ flux across isolated SR membrane vesicles using spectrophotometric and isotope methods. The health relatedness of the project is not only pertinent to malignant hyperthermia (linked to an abnormality in Ca2+ release from SR during anesthesia) but also more generally to numerous diseases of muscle weakness. The results of this investigation will additionally have health related implications for the cardiovascular and gastrointestinal systems, where cardiac and smooth muscle rely on Ca2+ release from SR for proper function. The results of this investigation may lead to suggestions for new drug therapies for cardiovascular disease.